The long-term goal of this project is to identify therapies that will preserve diaphragm function in chronic inflammatory conditions including heart failure and chronic obstructive pulmonary disease. Our current focus is loss of specific force in diaphragm which promotes exercise intolerance, breathlessness, and respiratory failure. A promising therapeutic target is tumor necrosis factor (TNF). TNF serum levels are elevated in chronic disease, correlate with muscle weakness, and are a predictor of morbidity and mortality. TNF depresses contractile function of diaphragm and stimulates oxidant production by diaphragm muscle fibers. The oxidants appear to cause weakness since interventions that limit oxidant activity prevent loss of force. Our central hypothesis is that respiratory muscle function can be preserved in chronic inflammatory disease by inhibiting TNF receptor-mediated effects on diaphragm muscle fibers. This project will define signaling events and redox mechanisms by which TNF depresses force and will evaluate pharmacologic and genetic interventions to preserve diaphragm function. We also will evaluate pathophysiologic relevance, defining the role of TNF in a mouse model of disease and testing the capacity of clinical therapeutic agents to preserve diaphragm function. The project addresses three specific aims: Aim 1. To evaluate sphingolipid signaling as an early post-receptor mechanism by which TNF/TNFR1 stimulates oxidant activity and weakness. Cell culture studies will assess sphingomyelinase activation and ceramide-sensitive signaling events downstream of the TNF receptor subtype 1. Aim 2. To assess the source, composition, and post-translational target of oxidants that mediate TNF-stimulated weakness. Diaphragm fiber bundles and isolated mitochondria will be used to define ROS and NO contributions via redox assays and pharmacologic and genetic tools. Aim 3. To test TNF signaling as a contributor to respiratory muscle weakness in heart failure and a potential target for therapy. An animal model of heart failure will be used to define the diaphragm response to chronic inflammation, to test TNF as a systemic mediator, and to evaluate potential therapies that are approved for use in humans.